Chlamydia antigens and corresponding DNA fragments and uses thereof

ABSTRACT

The present invention provides a method of nucleic acid, including DNA, immunization of a host, including humans, against disease caused by infection by a strain of  Chlamydia , specifically  C. pneumoniae , employing a vector containing a nucleotide sequence encoding a 60 kDa cysteine-rich membrane protein of a strain of  Chlamydia pneumoniae  and a promoter to effect expression of the 60 kDa cysteine-rich membrane protein gene in the host. Modifications are possible within the scope of this invention.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/123,966, filed Mar. 12, 1999.

FIELD OF INVENTION

The present invention relates to the Chlamydia 60 kDa cysteine-richmembrane protein and corresponding DNA molecules, which can be used toprevent and treat Chlamydia infection in mammals, such as humans.

BACKGROUND OF THE INVENTION

Chlamydiae are prokaryotes. They exhibit morphologic and structuralsimilarities to gram-negative bacteria including a trilaminar outermembrane, which contains lipopolysaccharide and several membraneproteins that are structurally and functionally analogous to proteinsfound in E coli. They are obligate intra-cellular parasites with aunique biphasic life cycle consisting of a metabolically inactive butinfectious extracellular stage and a replicating but non-infectiousintracellular stage. The replicative stage of the life-cycle takes placewithin a membrane-bound inclusion which sequesters the bacteria awayfrom the cytoplasm of the infected host cell.

C. pneumoniae is a common human pathogen, originally described as theTWAR strain of Chlamydia psittaci but subsequently recognised to be anew species. C. pneumoniae is antigenically, genetically andmorphologically distinct from other chlamydia species (C. trachomatis,C. pecorum and C. psittaci). It shows 10% or less DNA sequence homologywith either of C. trachomatis or C. psittaci.

C. pneumoniae is a common cause of community acquired pneumonia, onlyless frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae(Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Camposet al. (1995). Investigation of Ophthalmology and Visual Science36:1477). It can also cause upper respiratory tract symptoms anddisease, including bronchitis and sinusitis (Grayston et al. (1995)Journal of Infectious Diseases 168:1231; Grayston et al (1990) Journalof Infectious Diseases 161:618; Marrie (1993) Clinical InfectiousDiseases. 18:501; Wang et al (1986) Chlamydial infections CambridgeUniversity Press, Cambridge. p. 329. The great majority of the adultpopulation (over 60%) has antibodies to C. pneumoniae (Wang et al (1986)Chlamydial infections. Cambridge University Press, Cambridge. p. 329),indicating past infection which was unrecognized or asymptomatic.

C. pneumoniae infection usually presents as an acute respiratory disease(i.e., cough, sore throat, hoarseness, and fever; abnormal chest soundson auscultation). For most patients, the cough persists for 2 to 6weeks, and recovery is slow. In approximately 10% of these cases, upperrespiratory tract infection is followed by bronchitis or pneumonia.Furthermore, during a C. pneumoniae epidemic, subsequent co-infectionwith pneumococcus has been noted in about half of these pneumoniapatients, particularly in the infirm and the elderly. As noted above,there is more and more evidence that C. pneumoniae infection is alsolinked to diseases other than respiratory infections.

The reservoir for the organism is presumably people. In contrast to C.psittaci infections, there is no known bird or animal reservoir.Transmission has not been clearly defined. It may result from directcontact with secretions, from fomites, or from airborne spread. There isa long incubation period, which may last for many months. Based onanalysis of epidemics, C. pneumoniae appears to spread slowly through apopulation (case-to-case interval averaging 30 days) because infectedpersons are inefficient transmitters of the organism. Susceptibility toC. pneumoniae is universal. Reinfections occur during adulthood,following the primary infection as a child. C. pneumoniae appears to bean endemic disease throughout the world, noteworthy for superimposedintervals of increased incidence (epidemics) that persist for 2 to 3years. C. trachomatis infection does not confer cross-immunity to C.pneumoniae. Infections are easily treated with oral antibiotics,tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). Arecently developed drug, azithromycin, is highly effective as asingle-dose therapy against chlamydial infections.

In most instances, C. pneumoniae infection is often mild and withoutcomplications, and up to 90% of infections are subacute or unrecognized.Among children in industrialized countries, infections have been thoughtto be rare up to the age of 5 y, although a recent study (E Normann etal, Chlamydia pneumoniae in children with acute respiratory tractinfections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp 23-27) hasreported that many children in this age group show PCR evidence ofinfection despite being seronegative, and estimates a prevalence of17-19% in 2-4 y olds. In developing countries, the seroprevalence of C.pneumoniae antibodies among young children is elevated, and there aresuspicions that C. pneumoniae may be an important cause of acute lowerrespiratory tract disease and mortality for infants and children intropical regions of the world.

From seroprevalence studies and studies of local epidemics, the initialC. pneumoniae infection usually happens between the ages of 5 and 20 y.In the USA, for example, there are estimated to be 30,000 cases ofchildhood pneumonia each year caused by C. pneumoniae. Infections maycluster among groups of children or young adults (e.g., school pupils ormilitary conscripts).

C. pneumoniae causes 10 to 25% of community-acquired lower respiratorytract infections (as reported from Sweden, Italy, Finland, and the USA).During an epidemic, C. pneumonia infection may account for 50 to 60% ofthe cases of pneumonia. During these periods, also, more episodes ofmixed infections with S. pneumoniae have been reported.

Reinfection during adulthood is common; the clinical presentation tendsto be milder. Based on population seroprevalence studies, there tends tobe increased exposure with age, which is particularly evident among men.Some investigators have speculated that a persistent, asymptomatic C.pneumoniae infection state is common.

In adults of middle age or older, C. pneumoniae infection may progressto chronic bronchitis and sinusitis. A study in the USA revealed thatthe incidence of pneumonia caused by C. pneumoniae in persons youngerthan 60 years is 1 case per 1,000 persons per year; but in the elderly,the disease incidence rose three-fold. C. pneumoniae infection rarelyleads to hospitalization, except in patients with an underlying illness.

Of considerable importance is the association of atherosclerosis and C.pneumoniae infection. There are several epidemiological studies showinga correlation of previous infections with C. pneumoniae and heartattacks, coronary artery and carotid artery disease (Saikku et al.(1988) Lancet;ii:983; Thom et al. (1992) JAMA 268:68; Linnanmaki et al.(1993), Circulation 87:1030; Saikku et al. (1992) Annals InternalMedicine 116:273; Melnick et al(1993) American Journal of Medicine95:499). Moreover, the organisms has been detected in atheromas andfatty streaks of the coronary, carotid, peripheral arteries and aorta(Shor et al. (1992) South African. Medical Journal 82:158; Kuo et al.(1993) Journal of Infectious Diseases 167:841; Kuo et al. (1993)Arteriosclerosis and Thrombosis 13:1500; Campbell et al (1995) Journalof Infectious Diseases 172:585; Chiu et al. Circulation, 1997 (InPress)). Viable C. pneumoniae has been recovered from the coronary andcarotid artery (Ramirez et al (1996) Annals of Internal Medicine125:979; Jackson et al. Abst. K121, p272, 36^(th) ICAAC, 15-18 Sep.1996, New Orleans). Furthermore, it has been shown that C. pneumoniaecan induce changes of atherosclerosis in a rabbit model (Fong et al(1997) Journal of Clinical Microbiolology 35:48). Taken together, theseresults indicate that it is highly probable that C. pneumoniae can causeatherosclerosis in humans, though the epidemiological importance ofchlamydial atherosclerosis remains to be demonstrated.

A number of recent studies have also indicated an association between C.pneumoniae infection and asthma. Infection has been linked to wheezing,asthmatic bronchitis, adult-onset asthma and acute exacerbations ofasthma in adults, and small-scale studies have shown that prolongedantibiotic treatment was effective at greatly reducing the severity ofthe disease in some individuals (Hahn D L, et al. Evidence for Chlamydiapneumoniae infection in steroid-dependent asthma.Ann Allergy AsthmaImmunol. 1998 January; 80(1): 45-49.; Hahn D L, et al. Association ofChlamydia pneumoniae IgA antibodies with recently symptomatic asthma.Epidemiol Infect. 1996 December; 117(3): 513-517; Bjornsson E, et al.Serology of chlamydia in relation to asthma and bronchialhyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.; Hahn D L.Treatment of Chlamydia pneumoniae infection in adult asthma: abefore-after trial. J Fam Pract. 1995 October; 41(4): 345-351.; AllegraL, et al. Acute exacerbations of asthma in adults: role of Chlamydiapneumoniae infection. Eur Respir J. 1994 December; 7(12): 2165-2168.;Hahn DL, et al. Association of Chlamydia pneumoniae (strain TWAR)infection with wheezing, asthmatic bronchitis, and adult-onset asthma.JAMA. 1991 Jul. 10; 266(2): 225-230).

In light of these results a protective vaccine against C. pneumoniaeinfection would be of considerable importance. There is not yet aneffective vaccine for any human chlamydial infection. It is conceivablethat an effective vaccine can be developed using physically orchemically inactivated Chlamydiae. However, such a vaccine does not havea high margin of safety. In general, safer vaccines are made bygenetically manipulating the organism by attenuation or by recombinantmeans. Accordingly, a major obstacle in creating an effective and safevaccine against human chlamydial infection has been the paucity ofgenetic information regarding Chlamydia, specifically C. pneumoniae.

Studies with C. trachomatis and C. psittaci indicate that safe andeffective vaccine against Chlamydia is an attainable goal. For example,mice which have recovered from a lung infection with C. trachomatis areprotected from infertility induced by a subsequent vaginal challenge(Pal et al. (1996) Infection and Immunity.64:5341). Similarly, sheepimmunized with inactivated C. psittaci were protected from subsequentchlamydial-induced abortions and stillbirths (Jones et al. (1995)Vaccine 13:715). Protection from chlamydial infections has beenassociated with Th1 immune responses, particularly the induction ofINFg—producing CD4+ T-cells (Igietsemes et al. (1993) Immunology 5:317).The adoptive transfer of CD4+ cell lines or clones to nude or SCID miceconferred protection from challenge or cleared chronic disease(Igietseme et al (1993) Regional Immunology 5:317; Magee et al (1993)Regional Immunology 5: 305), and in vivo depletion of CD4+ T cellsexacerbated disease post-challenge (Landers et al (1991) Infection &Immunity 59:3774; Magee et al (1995) Infection & Immunity 63:516).However, the presence of sufficiently high titres of neutralisingantibody at mucosal surfaces can also exert a protective effect (Cotteret al. (1995) Infection and Immunity 63:4704).

Antigenic variation within the species C. pneumoniae is not welldocumented due to insufficient genetic information, though variation isexpected to exist based on C. trachomatis. Serovars of C. trachomatisare defined on the basis of antigenic variation in the major outermembrane protein (MOMP), but published C. pneumoniae MOMP gene sequencesshow no variation between several diverse isolates of the organism(Campbell et al (1990) Infection and Immunity 58:93; McCafferty et al(1995) Infection and Immunity 63:2387-9; Knudsen et al (1996) ThirdMeeting of the European Society for Chlamydia Research, Vienna). Thegene encoding a 76 kDa antigen has been cloned from a single strain ofC. pneumoniae and the sequence published (Perez Melgosa et al., Infect.Immun. 1994. 62:880). An operon encoding the 9 kDa and 60 kDacyteine-rich outer membrane protein genes has been described (Watson etal., Nucleic Acids Res (1990) 18:5299; Watson et al., Microbiology(1995) 141:2489). Many antigens recognized by immune sera to C.pneumoniae are conserved across all chlamydiae, but 98 kDa, 76 kDa andseveral other proteins may be C. pneumoniae-specific (Perez Melgosa etal., Infect. Immun. 1994. 62:880; Melgosa et al., FEMS Microbiol Lett(1993) 112:199;, Campbell et al., J Clin Microbiol (1990) 28:1261;Iijima et al., J Clin Microbiol (1994) 32:583). An assessment of thenumber and relative frequency of any C. pneumoniae serotypes, and thedefining antigens, is not yet possible. The entire genome sequence of C.pneumoniae strain CWL-029 is now known(http://chlamydia-www.berkeley.edu:4231/) and as further sequencesbecome available a better understanding of antigenic variation may begained.

Many antigens recognised by immune sera to C. pneumoniae are conservedacross all chlamydiae, but 98 kDa, 76 kDa and 54 kDa proteins appear tobe C. pneumoniae-specific (Campos et al. (1995) Investigation ofOphthalmology and Visual Science 36:1477; Marrie (1993) ClinicalInfectious Diseases. 18:501; Wiedmann-Al-Ahmad M, et al. Reactions ofpolyclonal and neutralizing anti-p54 monoclonal antibodies with anisolated, species-specific 54-kilodalton protein of Chlamydiapneumoniae. Clin Diagn Lab Immunol. 1997 November; 4(6): 700-704).

Immunoblotting of isolates with sera from patients does show variationof blotting patterns between isolates, indicating that serotypes C.pneumoniae may exist (Grayston et al. (1995) Journal of InfectiousDiseases 168:1231; Ramirez et al (1996) Annals of Internal Medicine125:979). However, the results are potentially confounded by theinfection status of the patients, since immunoblot profiles of apatient's sera change with time post-infection. An assessment of thenumber and relative frequency of any serotypes, and the definingantigens, is not yet possible.

Accordingly, a need exists for identifying and isolating polynucleotidesequences of C. pneumoniae for use in preventing and treating Chlamydiainfection.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated polynucleotidemolecules that encode the Chlamydia polypeptides designated 60 kDacysteine-rich membrane proteine (SEQ ID No:1) which can be used inmethods to prevent, treat, and diagnose Chlamydia infection. In one formof the invention, the polynucleotide molecules are DNA that encode thepolypeptide of SEQ ID No: 2.

Another form of the invention provides polypeptides corresponding to theisolated DNA molecules. The amino acid sequence of the correspondingencoded polypeptide is shown as SEQ ID No: 2.

Those skilled in the art will readily understand that the invention,having provided the polynucleotide sequences encoding the Chlamydia 60kDa cysteine-rich membrane protein, also provides polynucleotidesencoding fragments derived from such a polypeptide. Moreover, theinvention is understood to provide mutants and derivatives of suchpolypeptides and fragments derived therefrom, which result from theaddition, deletion, or substitution of non-essential amino acids asdescribed herein. Those skilled in the art would also readily understandthat the invention, having provided the polynucleotide sequencesencoding Chlamydia polypeptides, further provides monospecificantibodies that specifically bind to such polypeptides.

The present invention has wide application and includes expressioncassettes, vectors, and cells transformed or transfected with thepolynucleotides of the invention. Accordingly, the present inventionfurther provides (i) a method for producing a polypeptide of theinvention in a recombinant host system and related expression cassettes,vectors, and transformed or transfected cells; (ii) a vaccine, or a livevaccine vector such as a pox virus, Salmonella typhimurium, or Vibriocholerae vector, containing a polynucleotide of the invention, suchvaccines and vaccine vectors being useful for, e.g., preventing andtreating Chlamydia infection, in combination with a diluent or carrier,and related pharmaceutical compositions and associated therapeuticand/or prophylactic methods; (iii) a therapeutic and/or prophylactic useof an RNA or DNA molecule of the invention, either in a naked form orformulated with a delivery vehicle, a polypeptide or combination ofpolypeptides, or a monospecific antibody of the invention, and relatedpharmaceutical compositions; (iv) a method for diagnosing the presenceof Chlamydia in a biological sample, which can involve the use of a DNAor RNA molecule, a monospecific antibody, or a polypeptide of theinvention; and (v) a method for purifying a polypeptide of the inventionby antibody-based affinity chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the drawings, in which:

FIG. 1 shows the nucleotide sequence of the 60 kDa cysteine-richmembrane protein gene (SEQ ID No:1) and the deduced amino acid sequenceof the 60 kDa cysteine-rich membrane protein from Chlamydia pneumoniae(SEQ ID No: 2).

FIG. 2 shows the restriction enzyme analysis of the C. pneumoniae 60 kDacysteine-rich membrane protein gene.

FIG. 3 shows the construction and elements of plasmid pCACRMP60.

FIG. 4 illustrates protection against C. pneumoniae infection bypCACRMP60 following DNA immunization.

DETAILED DESCRIPTION OF INVENTION

An open reading frame (ORF) encoding the chlamydial 60 kDa cysteine-richmembrane protein has been identified from the C. pneumoniae genome. Thegene encoding this protein has been inserted into an expression plasmidand shown to confer immune protection against chlamydial infection.Accordingly, this 60 kDa cysteine-rich membrane protein and relatedpolypeptides can be used to prevent and treat Chlamydia infection.

According to a first aspect of the invention, isolated polynucleotidesare provided which encode Chlamydia polypeptides, whose amino acidsequences are shown in SEQ ID No: 2.

The term “isolated polynucleotide” is defined as a polynucleotideremoved from the environment in which it naturally occurs. For example,a naturally-occurring DNA molecule present in the genome of a livingbacteria or as part of a gene bank is not isolated, but the samemolecule separated from the remaining part of the bacterial genome, as aresult of, e.g., a cloning event (amplification), is isolated.Typically, an isolated DNA molecule is free from DNA regions (e.g.,coding regions) with which it is immediately contiguous at the 5′ or 3′end, in the naturally occurring genome. Such isolated polynucleotidesmay be part of a vector or a composition and still be defined asisolated in that such a vector or composition is not part of the naturalenvironment of such polynucleotide.

The polynucleotide of the invention is either RNA or DNA (cDNA, genomicDNA, or synthetic DNA), or modifications, variants, homologs orfragments thereof. The DNA is either double-stranded or single-stranded,and, if single-stranded, is either the coding strand or the non-coding(anti-sense) strand. Any one of the sequences that encode thepolypeptides of the invention as shown in SEQ ID No: 1 is (a) a codingsequence, (b) a ribonucleotide sequence derived from transcription of(a), or (c) a coding sequence which uses the redundancy or degeneracy ofthe genetic code to encode the same polypeptides. By “polypeptide” or“protein” is meant any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). Both terms are used interchangeably in the presentapplication.

Consistent with the first aspect of the invention, amino acid sequencesare provided which are homologous to SEQ ID No: 2. As used herein,“homologous amino acid sequence” is any polypeptide which is encoded, inwhole or in part, by a nucleic acid sequence which hybridizes at 25-35°C. below critical melting temperature (Tm), to any portion of thenucleic acid sequence of SEQ ID No: 1. A homologous amino acid sequenceis one that differs from an amino acid sequence shown in SEQ ID No: 2 byone or more conservative amino acid substitutions. Such a sequence alsoencompass serotypic variants (defined below) as well as sequencescontaining deletions or insertions which retain inherent characteristicsof the polypeptide such as immunogenicity. Preferably, such a sequenceis at least 75%, more preferably 80%, and most preferably 90% identicalto SEQ ID No: 2.

Homologous amino acid sequences include sequences that are identical orsubstantially identical to SEQ ID No: 2. By “amino acid sequencesubstantially identical” is meant a sequence that is at least 90%,preferably 95%, more preferably 97%, and most preferably 99% identicalto an amino acid sequence of reference and that preferably differs fromthe sequence of reference by a majority of conservative amino acidsubstitutions.

Conservative amino acid substitutions are substitutions among aminoacids of the same class. These classes include, for example, amino acidshaving uncharged polar side chains, such as asparagine, glutamine,serine, threonine, and tyrosine; amino acids having basic side chains,such as lysine, arginine, and histidine; amino acids having acidic sidechains, such as aspartic acid and glutamic acid; and amino acids havingnonpolar side chains, such as glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, andcysteine.

Homology is measured using sequence analysis software such as SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705. Amino acid sequences are aligned to maximize identity. Gaps maybe artificially introduced into the sequence to attain proper alignment.Once the optimal alignment has been set up, the degree of homology isestablished by recording all of the positions in which the amino acidsof both sequences are identical, relative to the total number ofpositions.

Homologous polynucleotide sequences are defined in a similar way.Preferably, a homologous sequence is one that is at least 45%, morepreferably 60%, and most preferably 85% identical to the coding sequenceof SEQ ID No: 1.

Consistent with the first aspect of the invention, polypeptides having asequence homologous to SEQ ID No: 2 include naturally-occurring allelicvariants, as well as mutants or any other non-naturally occurringvariants that retain the inherent characteristics of the polypeptide ofSEQ ID No: 2.

As is known in the art, an allelic variant is an alternate form of apolypeptide that is characterized as having a substitution, deletion, oraddition of one or more amino acids that does not alter the biologicalfunction of the polypeptide. By “biological function” is meant thefunction of the polypeptide in the cells in which it naturally occurs,even if the function is not necessary for the growth or survival of thecells. For example, the biological function of a porin is to allow theentry into cells of compounds present in the extracellular medium.Biological function is distinct from antigenic property. A polypeptidecan have more than one biological function.

Allelic variants are very common in nature. For example, a bacterialspecies such as C. pneumoniae, is usually represented by a variety ofstrains that differ from each other by minor allelic variations. Indeed,a polypeptide that fulfills the same biological function in differentstrains can have an amino acid sequence (and polynucleotide sequence)that is not identical in each of the strains. Despite this variation, animmune response directed generally against many allelic variants hasbeen demonstrated. In studies of the Chlamydial MOMP antigen,cross-strain antibody binding plus neutralization of infectivity occursdespite amino acid sequence variation of MOMP from strain to strain,indicating that the MOMP, when used as an immunogen, is tolerant ofamino acid variations.

Polynucleotides encoding homologous polypeptides or allelic variants areretrieved by polymerase chain reaction (PCR) amplification of genomicbacterial DNA extracted by conventional methods. This involves the useof synthetic oligonucleotide primers matching upstream and downstream ofthe 5′ and 3′ ends of the encoding domain. Suitable primers are designedaccording to the nucleotide sequence information provided in SEQ IDNo:1. The procedure is as follows: a primer is selected which consistsof 10 to 40, preferably 15 to 25 nucleotides. It is advantageous toselect primers containing C and G nucleotides in a proportion sufficientto ensure efficient hybridization; i.e., an amount of C and Gnucleotides of at least 40%, preferably 50% of the total nucleotidecontent. A standard PCR reaction contains typically 0.5 to 5 Units ofTaq DNA polymerase per 100 μL, 20 to 200 μM deoxynucleotide each,preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium overthe total deoxynucleotide concentration, 105 to 106 target molecules,and about 20 pmol of each primer. About 25 to 50 PCR cycles areperformed, with an annealing temperature 15° C. to 5° C. below the trueTm of the primers. A more stringent annealing temperature improvesdiscrimination against incorrectly annealed primers and reducesincorportion of incorrect nucleotides at the 3′ end of primers. Adenaturation temperature of 95° C. to 97° C. is typical, although highertemperatures may be appropriate for dematuration of G+C-rich targets.The number of cycles performed depends on the starting concentration oftarget molecules, though typically more than 40 cycles is notrecommended as non-specific background products tend to accumulate.

An alternative method for retrieving polynucleotides encoding homologouspolypeptides or allelic variants is by hybridization screening of a DNAor RNA library. Hybridization procedures are well-known in the art andare described in Ausubel et al., (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al.(Silhavy et al. Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, 1984), and Davis et al. (Davis et al. A Manual forGenetic Engineering: Advanced Bacterial Genetics, Cold Spring HarborLaboratory Press, 1980)). Important parameters for optimizinghybridization conditions are reflected in a formula used to obtain thecritical melting temperature above which two complementary DNA strandsseparate from each other (Casey & Davidson, Nucl. Acid Res. (1977)4:1539). For polynucleotides of about 600 nucleotides or larger, thisformula is as follows: Tm=81.5+0.41×(% G+C)+16.6 log (cation ionconcentration)-0.63×(% formamide)-600/base number. Under appropriatestringency conditions, hybridization temperature (Th) is approximately20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C. below thecalculated Tm. Those skilled in the art will understand that optimaltemperature and salt conditions can be readily determined.

For the polynucleotides of the invention, stringent conditions areachieved for both pre-hybridizing and hybridizing incubations (i) within4-16 hours at 42° C., in 6×SSC containing 50% formamide, or (ii) within4-16 hours at 65° C. in an aqueous 6×SSC solution (1 M NaCl, 0.1 Msodium citrate (pH 7.0)). Typically, hybridization experiments areperformed at a temperature from 60 to 68° C., e.g. 65° C. At such atemperature, stringent hybridization conditions can be achieved in6×SSC, preferably in 2×SSC or 1×SSC, more preferably in 0.5×SSc, 0.3×SSCor 0.1×SSC (in the absence of formamide). 1×SSC contains 0.15 M NaCl and0015 M sodium citrate.

Useful homologs and fragments thereof that do not occur naturally aredesigned using known methods for identifying regions of an antigen thatare likely to tolerate amino acid sequence changes and/or deletions. Asan example, homologous polypeptides from different species are compared;conserved sequences are identified. The more divergent sequences are themost likely to tolerate sequence changes. Homology among sequences maybe analyzed using, as an example, the BLAST homology searching algorithmof Altschul et al., Nucleic Acids Res.; 25:3389-3402 (1997).Alternatively, sequences are modified such that they become morereactive to T- and/or B-cells, based on computer-assisted analysis ofprobable T- or B-cell epitopes Yet another alternative is to mutate aparticular amino acid residue or sequence within the polypeptide invitro, then screen the mutant polypeptides for their ability to preventor treat Chlamydia infection according to the method outlined below.

A person skilled in the art will readily understand that by followingthe screening process of this invention, it will be determined withoutundue experimentation whether a particular homolog of SEQ ID No. 2 maybe useful in the prevention or treatment of Chlamydia infection. Thescreening procedure comprises the steps:

-   -   (i) immunizing an animal, preferably mouse, with the test        homolog or fragment;    -   (ii) inoculating the immunized animal with Chlamydia; and    -   (iii) selecting those homologs or fragments which confer        protection against Chlamydia.

By “conferring protection” is meant that there is a reduction inseverity of any of the effects of Chlamydia infection, in comparisonwith a control animal which was not immunized with the test homolog orfragment.

Consistent with the first aspect of the invention, polypeptidederivatives are provided that are partial sequences of SEQ ID No. 2,partial sequences of polypeptide sequences homologous to SEQ ID No. 2,polypeptides derived from full-length polypeptides by internal deletion,and fusion proteins.

It is an accepted practice in the field of immunology to use fragmentsand variants of protein immunogens as vaccines, as all that is requiredto induce an immune response to a protein is a small (e.g., 8 to 10amino acid) immunogenic region of the protein. Various short syntheticpeptides corresponding to surface-exposed antigens of pathogens otherthan Chlamydia have been shown to be effective vaccine antigens againsttheir respective pathogens, e.g. an 11 residue peptide of murine mammarytumor virus (Casey & Davidson, Nucl. Acid Res. (1977) 4:1539), a16-residue peptide of Semliki Forest virus (Snijders et al., 1991. J.Gen. Virol. 72:557-565), and two overlapping peptides of 15 residueseach from canine parvovirus (Langeveld et al., Vaccine 12(15):1473-1480,1994).

Accordingly, it will be readily apparent to one skilled in the art,having read the present description, that partial sequences of SEQ IDNo: 2 or their homologous amino acid sequences are inherent to thefull-length sequences and are taught by the present invention. Suchpolypeptide fragments preferably are at least 12 amino acids in length.Advantageously, they are at least 20 amino acids, preferably at least 50amino acids, more preferably at least 75 amino acids, and mostpreferably at least 100 amino acids in length.

Polynucleotides of 30 to 600 nucleotides encoding partial sequences ofsequences homologous to SEQ ID No: 2 are retrieved by PCR amplificationusing the parameters outlined above and using primers matching thesequences upstream and downstream of the 5′ and 3′ ends of the fragmentto be amplified. The template polynucleotide for such amplification iseither the full length polynucleotide homologous to SEQ ID No:1, or apolynucleotide contained in a mixture of polynucleotides such as a DNAor RNA library. As an alternative method for retrieving the partialsequences, screening hybridization is carried out under conditionsdescribed above and using the formula for calculating Tm. Wherefragments of 30 to 600 nucleotides are to be retrieved, the calculatedTm is corrected by subtracting (600/polynucleotide size in base pairs)and the stringency conditions are defined by a hybridization temperaturethat is 5 to 10° C. below Tm. Where oligonucleotides shorter than 20-30bases are to be obtained, the formula for calculating the Tm is asfollows: Tm=4×(G+C)+2 (A+T). For example, an 18 nucleotide fragment of50% G+C would have an approximate Tm of 54° C. Short peptides that arefragments of SEQ ID No: 2 or its homologous sequences, are obtaineddirectly by chemical synthesis (E. Gross and H. J. Meinhofer, 4 ThePeptides: Analysis, Synthesis, Biology; Modern Techniques of PeptideSynthesis, John Wiley & Sons (1981), and M. Bodanzki, Principles ofPeptide Synthesis, Springer-Verlag (1984)).

Useful polypeptide derivatives, e.g., polypeptide fragments, aredesigned using computer-assisted analysis of amino acid sequences. Thiswould identify probable surface-exposed, antigenic regions (Hughes etal., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acidsequences contained in SEQ ID No: 2, based on the product of flexibilityand hydrophobicity propensities using the program SEQSEE (Wishart DS, etal. “SEQSEE: a comprehensive program suite for protein sequenceanalysis.” Comput Appl Biosci. 1994 April;10(2):121-32), can revealpotential B- and T-cell epitopes which may be used as a basis forselecting useful immunogenic fragments and variants. This analysis usesa reasonable combination of external surface features that is likely tobe recognized by antibodies. Probable T-cell epitopes for HLA-A0201 MHCsubclass may be revealed by an algorithms that emulate an approachdeveloped at the NIH (Parker KC, et al. “Peptide binding to MHC class Imolecules: implications for antigenic peptide prediction.” Immunol Res1995;14(1):34-57).

Epitopes which induce a protective T cell-dependent immune response arepresent throughout the length of the polypeptide. However, some epitopesmay be masked by secondary and tertiary structures of the polypeptide.To reveal such masked epitopes large internal deletions are createdwhich remove much of the original protein structure and exposes themasked epitopes. Such internal deletions sometimes effect the additionaladvantage of removing immunodominant regions of high variability amongstrains.

Polynucleotides encoding polypeptide fragments and polypeptides havinglarge internal deletions are constructed using standard methods (Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons Inc.,1994). Such methods include standard PCR, inverse PCR, restrictionenzyme treatment of cloned DNA molecules, or the method of Kunkel et al.(Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components forthese methods and instructions for their use are readily available fromvarious commercial sources such as Stratagene. Once the deletion mutantshave been constructed, they are tested for their ability to prevent ortreat Chlamydia infection as described above.

As used herein, a fusion polypeptide is one that contains a polypeptideor a polypeptide derivative of the invention fused at the N- orC-terminal end to any other polypeptide (hereinafter referred to as apeptide tail). A simple way to obtain such a fusion polypeptide is bytranslation of an in-frame fusion of the polynucleotide sequences, i.e.,a hybrid gene. The hybrid gene encoding the fusion polypeptide isinserted into an expression vector which is used to transform ortransfect a host cell. Alternatively, the polynucleotide sequenceencoding the polypeptide or polypeptide derivative is inserted into anexpression vector in which the polynucleotide encoding the peptide tailis already present. Such vectors and instructions for their use arecommercially available, e.g. the pMal-c2 or pMal-p2 system from NewEngland Biolabs, in which the peptide tail is a maltose binding protein,the glutathione-s-transferase system of Pharmacia, or the His-Tag systemavailable from Novagen. These and other expression systems provideconvenient means for further purification of polypeptides andderivatives of the invention.

An advantageous example of a fusion polypeptide is one where thepolypeptide or homolog or fragment of the invention is fused to apolypeptide having adjuvant activity, such as subunit B of eithercholera toxin or E. coli heat-labile toxin. Another advantageous fusionis one where the polypeptide, homolog or fragment is fused to a strongT-cell epitope or B-cell epitope. Such an epitope may be one known inthe art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al.,“Antibody production to the nucleocapsid and envelope of the Hepatitis Bvirus primed by a single synthetic T cell site”, Nature. 1987.329:547-549), or one which has been identified in another polypeptide ofthe invention based on computer-assisted analysis of probable T- orB-cell epitopes. Consistent with this aspect of the invention is afusion polypeptide comprising T- or B-cell epitopes from SEQ ID No: 2 orits homolog or fragment, wherein the epitopes are derived from multiplevariants of said polypeptide or homolog or fragment, each variantdiffering from another in the location and sequence of its epitopewithin the polypeptide. Such a fusion is effective in the prevention andtreatment of Chlamydia infection since it optimizes the T- and B-cellresponse to the overall polypeptide, homolog or fragment.

To effect fusion, the polypeptide of the invention is fused to the N-,or preferably, to the C-terminal end of the polypeptide having adjuvantactivity or T- or B-cell epitope. Alternatively, a polypeptide fragmentof the invention is inserted internally within the amino acid sequenceof the polypeptide having adjuvant activity. The T- or B-cell epitopemay also be inserted internally within the amino acid sequence of thepolypeptide of the invention.

Consistent with the first aspect, the polynucleotides of the inventionalso encode hybrid precursor polypeptides containing heterologous signalpeptides, which mature into polypeptides of the invention. By“heterologous signal peptide” is meant a signal peptide that is notfound in naturally-occurring precursors of polypeptides of theinvention.

Polynucleotide molecules according to the invention, including RNA, DNA,or modifications or combinations thereof, have various applications. ADNA molecule is used, for example, (i) in a process for producing theencoded polypeptide in a recombinant host system, (ii) in theconstruction of vaccine vectors such as poxviruses, which are furtherused in methods and compositions for preventing and/or treatingChlamydia infection, (iii) as a vaccine agent (as well as an RNAmolecule), in a naked form or formulated with a delivery vehicle and,(iv) in the construction of attenuated Chlamydia strains that canover-express a polynucleotide of the invention or express it in anon-toxic, mutated form.

Accordingly, a second aspect of the invention encompasses (i) anexpression cassette containing a DNA molecule of the invention placedunder the control of the elements required for expression, in particularunder the control of an appropriate promoter; (ii) an expression vectorcontaining an expression cassette of the invention; (iii) a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, as well as (iv) a process forproducing a polypeptide or polypeptide derivative encoded by apolynucleotide of the invention, which involves culturing a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, under conditions that allowexpression of the DNA molecule of the invention and, recovering theencoded polypeptide or polypeptide derivative from the cell culture.

A recombinant expression system is selected from procaryotic andeucaryotic hosts. Eucaryotic hosts include yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g.,COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodopterafrugiperda (SF9) cells), and plant cells. A preferred expression systemis a procaryotic host such as E. coli. Bacterial and eucaryotic cellsare available from a number of different sources including commercialsources to those skilled in the art, e.g., the American Type CultureCollection (ATCC; Rockville, Md.). Commercial sources of cells used forrecombinant protein expression also provide instructions for usage ofthe cells.

The choice of the expression system depends on the features desired forthe expressed polypeptide. For example, it may be useful to produce apolypeptide of the invention in a particular lipidated form or any otherform.

One skilled in the art would redily understand that not all vectors andexpression control sequences and hosts would be expected to expressequally well the polynucleotides of this invention. With the guidelinesdescribed below, however, a selection of vectors, expression controlsequences and hosts may be made without undue experimentation andwithout departing from the scope of this invention.

In selecting a vector, the host must be chosen that is compatible withthe vector which is to exist and possibly replicate in it.Considerations are made with respect to the vector copy number, theability to control the copy number, expression of other proteins such asantibiotic resistance. In selecting an expression control sequence, anumber of variables are considered. Among the important variable are therelative strength of the sequence (e.g. the ability to drive expressionunder various conditions), the ability to control the sequence'sfunction, compatibility between the polynucleotide to be expressed andthe control sequence (e.g. secondary structures are considered to avoidhairpin structures which prevent efficient transcription). In selectingthe host, unicellular hosts are selected which are compatible with theselected vector, tolerant of any possible toxic effects of the expressedproduct, able to secrete the expressed product efficiently if such isdesired, to be able to express the product in the desired conformation,to be easily scaled up, and to which ease of purification of the finalproduct.

The choice of the expression cassette depends on the host systemselected as well as the features desired for the expressed polypeptide.Typically, an expression cassette includes a promoter that is functionalin the selected host system and can be constitutive or inducible; aribosome binding site; a start codon (ATG) if necessary; a regionencoding a signal peptide, e.g., a lipidation signal peptide; a DNAmolecule of the invention; a stop codon; and optionally a 3′ terminalregion (translation and/or transcription terminator) The signal peptideencoding region is adjacent to the polynucleotide of the invention andplaced in proper reading frame. The signal peptide-encoding region ishomologous or heterologous to the DNA molecule encoding the maturepolypeptide and is compatible with the secretion apparatus of the hostused for expression. The open reading frame constituted by the DNAmolecule of the invention, solely or together with the signal peptide,is placed under the control of the promoter so that transcription andtranslation occur in the host system. Promoters and signal peptideencoding regions are widely known and available to those skilled in theart and include, for example, the promoter of Salmonella typhimurium(and derivatives) that is inducible by arabinose (promoter araB) and isfunctional in Gram-negative bacteria such as E. coli (as described inU.S. Pat. No. 5,028,530 and in Cagnon et al., (Cagnon et al., ProteinEngineering (1991) 4(7):843)); the promoter of the gene of bacteriophageT7 encoding RNA polymerase, that is functional in a number of E. colistrains expressing T7 polymerase (described in U.S. Pat. No. 4,952,496);OspA lipidation signal peptide; and RlpB lipidation signal peptide(Takase et al., J. Bact. (1987) 169:5692).

The expression cassette is typically part of an expression vector, whichis selected for its ability to replicate in the chosen expressionsystem. Expression vectors (e.g., plasmids or viral vectors) can bechosen, for example, from those described in Pouwels et al. (CloningVectors: A Laboratory Manual 1985, Supp. 1987). Suitable expressionvectors can be purchased from various commercial sources.

Methods for transforming/transfecting host cells with expression vectorsare well-known in the art and depend on the host system selected asdescribed in Ausubel et al., (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc., 1994).

Upon expression, a recombinant polypeptide of the invention (or apolypeptide derivative) is produced and remains in the intracellularcompartment, is secreted/excreted in the extracellular medium or in theperiplasmic space, or is embedded in the cellular membrane. Thepolypeptide is recovered in a substantially purified form from the cellextract or from the supernatant after centrifugation of the recombinantcell culture. Typically, the recombinant polypeptide is purified byantibody-based affinity purification or by other well-known methods thatcan be readily adapted by a person skilled in the art, such as fusion ofthe polynucleotide encoding the polypeptide or its derivative to a smallaffinity binding domain. Antibodies useful for purifying byimmunoaffinity the polypeptides of the invention are obtained asdescribed below.

A polynucleotide of the invention can also be useful as a vaccine. Thereare two major routes, either using a viral or bacterial host as genedelivery vehicle (live vaccine vector) or administering the gene in afree form, e.g., inserted into a plasmid. Therapeutic or prophylacticefficacy of a polynucleotide of the invention is evaluated as describedbelow.

Accordingly, a third aspect of the invention provides (i) a vaccinevector such as a poxvirus, containing a DNA molecule of the invention,placed under the control of elements required for expression; (ii) acomposition of matter comprising a vaccine vector of the invention,together with a diluent or carrier; specifically (iii) a pharmaceuticalcomposition containing a therapeutically or prophylactically effectiveamount of a vaccine vector of the invention; (iv) a method for inducingan immune response against Chlamydia in a mammal (e.g., a human;alternatively, the method can be used in veterinary applications fortreating or preventing Chlamydia infection of animals, e.g., cats orbirds), which involves administering to the mammal an immunogenicallyeffective amount of a vaccine vector of the invention to elicit aprotective or therapeutic immune response to Chlamydia; andparticularly, (v) a method for preventing and/or treating a Chlamydia(e.g., C. trachomatis, C. psittaci, C. pneumonia, C. pecorum) infection,which involves administering a prophylactic or therapeutic amount of avaccine vector of the invention to an infected individual. Additionally,the third aspect of the invention encompasses the use of a vaccinevector of the invention in the preparation of a medicament forpreventing and/or treating Chlamydia infection.

As used herein, a vaccine vector expresses one or several polypeptidesor derivatives of the invention. The vaccine vector may expressadditionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12(IL-12), that enhances the immune response (adjuvant effect). It isunderstood that each of the components to be expressed is placed underthe control of elements required for expression in a mammalian cell.

Consistent with the third aspect of the invention is a compositioncomprising several vaccine vectors, each of them capable of expressing apolypeptide or derivative of the invention. A composition may alsocomprise a vaccine vector capable of expressing an additional Chlamydiaantigen, or a subunit, fragment, homolog, mutant, or derivative thereof;optionally together with or a cytokine such as IL-2 or IL-12.

Vaccination methods for treating or preventing infection in a mammalcomprises use of a vaccine vector of the invention to be administered byany conventional route, particularly to a mucosal (e.g., ocular,intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, orurinary tract) surface or via the parenteral (e.g., subcutaneous,intradermal, intramuscular, intravenous, or intraperitoneal) route.Preferred routes depend upon the choice of the vaccine vector. Treatmentmay be effected in a single dose or repeated at intervals. Theappropriate dosage depends on various parameters understood by skilledartisans such as the vaccine vector itself, the route of administrationor the condition of the mammal to be vaccinated (weight, age and thelike).

Live vaccine vectors available in the art include viral vectors such asadenoviruses and poxviruses as well as bacterial vectors, e.g.,Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié deCalmette-Guérin (BCG), and Streptococcus.

An example of an adenovirus vector, as well as a method for constructingan adenovirus vector capable of expressing a DNA molecule of theinvention, are described in U.S. Pat. No. 4,920,209. Poxvirus vectorsinclude vaccinia and canary pox virus, described in U.S. Pat. No.4,722,848 and U.S. Pat. No. 5,364,773, respectively. (Also see, e.g.,Tartaglia et al., Virology (1992) 188:217) for a description of avaccinia virus vector and Taylor et al, Vaccine (1995) 13:539 for areference of a canary pox.) Poxvirus vectors capable of expressing apolynucleotide of the invention are obtained by homologous recombinationas described in Kieny et al., Nature (1984) 312:163 so that thepolynucleotide of the invention is inserted in the viral genome underappropriate conditions for expression in mammalian cells. Generally, thedose of vaccine viral vector, for therapeutic or prophylactic use, canbe of from about 1×10⁴ to about 1×10¹¹, advantageously from about 1×10⁷to about 1×10¹⁰, preferably of from about 1×10⁷ to about 1×10⁹plaque-forming units per kilogram. Preferably, viral vectors areadministered parenterally; for example, in 3 doses, 4 weeks apart. It ispreferable to avoid adding a chemical adjuvant to a compositioncontaining a viral vector of the invention and thereby minimizing theimmune response to the viral vector itself.

Non-toxicogenic Vibrio cholerae mutant strains that are useful as a liveoral vaccine are known. Mekalanos et al., Nature (1983) 306:551 and U.S.Pat. No. 4,882,278 describe strains which have a substantial amount ofthe coding sequence of each of the two ctxA alleles deleted so that nofunctional cholerae toxin is produced. WO 92/11354 describes a strain inwhich the irgA locus is inactivated by mutation; this mutation can becombined in a single strain with ctxA mutations. WO 94/01533 describes adeletion mutant lacking functional ctxA and attRS1 DNA sequences. Thesemutant strains are genetically engineered to express heterologousantigens, as described in WO 94/19482. An effective vaccine dose of aVibrio cholerae strain capable of expressing a polypeptide orpolypeptide derivative encoded by a DNA molecule of the inventioncontains about 1×10⁵ to about 1×10⁹, preferably about 1×10⁶ to about1×10⁸, viable bacteria in a volume appropriate for the selected route ofadministration. Preferred routes of administration include all mucosalroutes; most preferably, these vectors are administered intranasally ororally.

Attenuated Salmonella typhimurium strains, genetically engineered forrecombinant expression of heterologous antigens or not, and their use asoral vaccines are described in Nakayama et al. (Bio/Technology (1988)6:693) and WO 92/11361. Preferred routes of administration include allmucosal routes; most preferably, these vectors are administeredintranasally or orally.

Other bacterial strains used as vaccine vectors in the context of thepresent invention are described for Shigella flexneri in High et al.,EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299; forStreptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci. USA(1995) 92:6868; and for Bacille Calmette Guerin in Flynn J. L., Cell.Mol. Biol. (1994) 40 (suppl. I):31, WO 88/06626, WO 90/00594, WO91/13157, WO 92/01796, and WO 92/21376.

In bacterial vectors, the polynucleotide of the invention is insertedinto the bacterial genome or remains in a free state as part of aplasmid.

The composition comprising a vaccine bacterial vector of the presentinvention may further contain an adjuvant. A number of adjuvants areknown to those skilled in the art. Preferred adjuvants are selected asprovided below.

Accordingly, a fourth aspect of the invention provides (i) a compositionof matter comprising a polynucleotide of the invention, together with adiluent or carrier; (ii) a pharmaceutical composition comprising atherapeutically or prophylactically effective amount of a polynucleotideof the invention; (iii) a method for inducing an immune response againstChlamydia in a mammal by administration of an immunogenically effectiveamount of a polynucleotide of the invention to elicit a protectiveimmune response to Chlamydia; and particularly, (iv) a method forpreventing and/or treating a Chlamydia (e.g., C. trachomatis, C.psittaci, C. pneumoniae, or C. pecorum) infection, by administering aprophylactic or therapeutic amount of a polynucleotide of the inventionto an infected individual. Additionally, the fourth aspect of theinvention encompasses the use of a polynucleotide of the invention inthe preparation of a medicament for preventing and/or treating Chlamydiainfection. A preferred use includes the use of a DNA molecule placedunder conditions for expression in a mammalian cell, especially in aplasmid that is unable to replicate in mammalian cells and tosubstantially integrate in a mammalian genome.

Use of the polynucleotides of the invention include their administrationto a mammal as a vaccine, for therapeutic or prophylactic purposes. Suchpolynucleotides are used in the form of DNA as part of a plasmid that isunable to replicate in a mammalian cell and unable to integrate into themammalian genome. Typically, such a DNA molecule is placed under thecontrol of a promoter suitable for expression in a mammalian cell. Thepromoter functions either ubiquitously or tissue-specifically. Examplesof non-tissue specific promoters include the early Cytomegalovirus (CMV)promoter (described in U.S. Pat. No. 4,168,062) and the Rous SarcomaVirus promoter (described in Norton & Coffin, Molec. Cell Biol. (1985)5:281). An example of a tissue-specific promoter is the desmin promoterwhich drives expression in muscle cells (Li et al., Gene (1989) 78:243,Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol.Chem. (1993) 268:10403). Use of promoters is well-known to those skilledin the art. Useful vectors are described in numerous publications,specifically WO 94/21797 and Hartikka et al., Human Gene Therapy (1996)7:1205.

Polynucleotides of the invention which are used as vaccines encodeeither a precursor or a mature form of the corresponding polypeptide. Inthe precursor form, the signal peptide is either homologous orheterologous. In the latter case, a eucaryotic leader sequence such asthe leader sequence of the tissue-type plasminogen factor (tPA) ispreferred.

As used herein, a composition of the invention contains one or severalpolynucleotides with optionally at least one additional polynucleotideencoding another Chlamydia antigen such as urease subunit A, B, or both,or a fragment, derivative, mutant, or analog thereof. The compositionmay also contain an additional polynucleotide encoding a cytokine, suchas interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immuneresponse is enhanced. These additional polynucleotides are placed underappropriate control for expression. Advantageously, DNA molecules of theinvention and/or additional DNA molecules to be included in the samecomposition, are present in the same plasmid.

Standard techniques of molecular biology for preparing and purifyingpolynucleotides are used in the preparation of polynucleotidetherapeutics of the invention. For use as a vaccine, a polynucleotide ofthe invention is formulated according to various methods outlined below.

One method utililizes the polynucleotide in a naked form, free of anydelivery vehicles. Such a polynucleotide is simply diluted in aphysiologically acceptable solution such as sterile saline or sterilebuffered saline, with or without a carrier. When present, the carrierpreferably is isotonic, hypotonic, or weakly hypertonic, and has arelatively low ionic strength, such as provided by a sucrose solution,e.g., a solution containing 20% sucrose.

An alternative method utilizes the polynucleotide in association withagents that assist in cellular uptake. Examples of such agents are (i)chemicals that modify cellular permeability, such as bupivacaine (see,e.g., WO 94/16737), (ii) liposomes for encapsulation of thepolynucleotide, or (iii) cationic lipids or silica, gold, or tungstenmicroparticles which associate themselves with the polynucleotides.

Anionic and neutral liposomes are well-known in the art (see, e.g.,Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for adetailed description of methods for making liposomes) and are useful fordelivering a large range of products, including polynucleotides.

Cationic lipids are also known in the art and are commonly used for genedelivery. Such lipids include Lipofectin™ also known as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio) propane), DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine), as described in WO 90/11092 as an example.

Formulation containing cationic liposomes may optionally contain othertransfection-facilitating compounds. A number of them are described inWO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They includespermine derivatives useful for facilitating the transport of DNAthrough the nuclear membrane (see, for example, WO 93/18759) andmembrane-permeabilizing compounds such as GALA, Gramicidine S, andcationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles are used for gene delivery, as describedin WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152. Themicroparticle-coated polynucleotide is injected via intradermal orintraepidermal routes using a needleless injection device (“gene gun”),such as those described in U.S. Pat. No. 4,945,050, U.S. Pat. No.5,015,580, and WO 94/24263.

The amount of DNA to be used in a vaccine recipient depends, e.g., onthe strength of the promoter used in the DNA construct, theimmunogenicity of the expressed gene product, the condition of themammal intended for administration (e.g., the weight, age, and generalhealth of the mammal), the mode of administration, and the type offormulation. In general, a therapeutically or prophylactically effectivedose from about 1 μg to about 1 mg, preferably, from about 10 μg toabout 800 μg and, more preferably, from about 25 μg to about 250 μg, canbe administered to human adults. The administration can be achieved in asingle dose or repeated at intervals.

The route of administration is any conventional route used in thevaccine field. As general guidance, a polynucleotide of the invention isadministered via a mucosal surface, e.g., an ocular, intranasal,pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface;or via a parenteral route, e.g., by an intravenous, subcutaneous,intraperitoneal, intradermal, intraepidermal, or intramuscular route.The choice of administration route depends on the formulation that isselected. A polynucleotide formulated in association with bupivacaine isadvantageously administered into muscles. When a neutral or anionicliposome or a cationic lipid, such as DOTMA or DC-Chol, is used, theformulation can be advantageously injected via intravenous, intranasal(aerosolization), intramuscular, intradermal, and subcutaneous routes. Apolynucleotide in a naked form can advantageously be administered viathe intramuscular, intradermal, or sub-cutaneous routes.

Although not absolutely required, such a composition can also contain anadjuvant. If so, a systemic adjuvant that does not require concomitantadministration in order to exhibit an adjuvant effect is preferable suchas, e.g., QS21, which is described in U.S. Pat. No. 5,057,546.

The sequence information provided in the present application enables thedesign of specific nucleotide probes and primers that are used fordiagnostic purposes. Accordingly, a fifth aspect of the inventionprovides a nucleotide probe or primer having a sequence found in orderived by degeneracy of the genetic code from a sequence shown in SEQID No:1.

The term “probe” as used in the present application refers to DNA(preferably single stranded) or RNA molecules (or modifications orcombinations thereof) that hybridize under the stringent conditions, asdefined above, to nucleic acid molecules having SEQ ID No:1 or tosequences homologous to SEQ ID No:1, or to its complementary oranti-sense sequence. Generally, probes are significantly shorter thanfull-length sequences. Such probes contain from about 5 to about 100,preferably from about 10 to about 80, nucleotides. In particular, probeshave sequences that are at least 75%, preferably at least 85%, morepreferably 95% homologous to a portion of SEQ ID No:1 or that arecomplementary to such sequences. Probes may contain modified bases suchas inosine, methyl-5-deoxycytidine, deoxyuridine,dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphateresidues may also be modified or substituted. For example, a deoxyriboseresidue may be replaced by a polyamide (Nielsen et al., Science (1991)254:1497) and phosphate residues may be replaced by ester groups such asdiphosphate, alkyl, arylphosphonate and phosphorothioate esters. Inaddition, the 2′-hydroxyl group on ribonucleotides may be modified byincluding such groups as alkyl groups.

Probes of the invention are used in diagnostic tests, as capture ordetection probes. Such capture probes are conventionally immobilized ona solid support, directly or indirectly, by covalent means or by passiveadsorption. A detection probe is labelled by a detection marker selectedfrom: radioactive isotopes, enzymes such as peroxidase, alkalinephosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic,or luminescent substrate, compounds that are chromogenic, fluorogenic,or luminescent, nucleotide base analogs, and biotin.

Probes of the invention are used in any conventional hybridizationtechnique, such as dot blot (Maniatis et al., Molecular Cloning: ALaboratory Manual (1982) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975)98:503), northern blot (identical to Southern blot with the exceptionthat RNA is used as a target), or the sandwich technique (Dunn et al.,Cell (1977) 12:23). The latter technique involves the use of a specificcapture probe and/or a specific detection probe with nucleotidesequences that at least partially differ from each other.

A primer is a probe of usually about 10 to about 40 nucleotides that isused to initiate enzymatic polymerization of DNA in an amplificationprocess (e.g., PCR), in an elongation process, or in a reversetranscription method. Primers used in diagnostic methods involving PCRare labeled by methods known in the art.

As described herein, the invention also encompasses (i) a reagentcomprising a probe of the invention for detecting and/or identifying thepresence of Chlamydia in a biological material; (ii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA or RNA is extracted from the material anddenatured, and (c) exposed to a probe of the invention, for example, acapture, detection probe or both, under stringent hybridizationconditions, such that hybridization is detected; and (iii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA is extracted therefrom, (c) the extractedDNA is primed with at least one, and preferably two, primers of theinvention and amplified by polymerase chain reaction, and (d) theamplified DNA fragment is produced.

It is apparent that disclosure of polynucleotide sequences of SEQ IDNo:1, its homologs and partial sequences enable their correspondingamino acid sequences. Accordingly, a sixth aspect of the inventionfeatures a substantially purified polypeptide or polypeptide derivativehaving an amino acid sequence encoded by a polynucleotide of theinvention.

A “substantially purified polypeptide” as used herein is defined as apolypeptide that is separated from the environment in which it naturallyoccurs and/or that is free of the majority of the polypeptides that arepresent in the environment in which it was synthesized. For example, asubstantially purified polypeptide is free from cytoplasmicpolypeptides. Those skilled in the art would readily understand that thepolypeptides of the invention may be purified from a natural source,i.e., a Chlamydia strain, or produced by recombinant means.

Consistent with the sixth aspect of the invention are polypeptides,homologs or fragments which are modified or treated to enhance theirimmunogenicity in the target animal, in whom the polypeptide, homolog orfragments are intended to confer protection against Chlamydia. Suchmodifications or treatments include: amino acid substitutions with anamino acid derivative such as 3-methyhistidine, 4-hydroxyproline,5-hydroxylysine etc., modifications or deletions which are carried outafter preparation of the polypeptide, homolog or fragment, such as themodification of free amino, carboxyl or hydroxyl side groups of theamino acids.

Identification of homologous polypeptides or polypeptide derivativesencoded by polynucleotides of the invention which have specificantigenicity is achieved by screening for cross-reactivity with anantiserum raised against the polypeptide of reference having an aminoacid sequence of SEQ ID No:1. The procedure is as follows: amonospecific hyperimmune antiserum is raised against a purifiedreference polypeptide, a fusion polypeptide (for example, an expressionproduct of MBP, GST, or His-tag systems, the description andinstructions for use of which are contained in Invitrogen productmanuals for pcDNA3.1/Myc-His(+) A, B, and C and for the Xpress™ SystemProtein Purification), or a synthetic peptide predicted to be antigenic.Where an antiserum is raised against a fusion polypeptide, two differentfusion systems are employed. Specific antigenicity can be determinedaccording to a number of methods, including Western blot (Towbin et al.,Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, asdescribed below.

In a Western blot assay, the product to be screened, either as apurified preparation or a total E. coli extract, is submitted toSDS-Page electrophoresis as described by Laemmli (Nature (1970)227:680). After transfer to a nitrocellulose membrane, the material isfurther incubated with the monospecific hyperimmune antiserum diluted inthe range of dilutions from about 1:5 to about 1:5000, preferably fromabout 1:100 to about 1:500. Specific antigenicity is shown once a bandcorresponding to the product exhibits reactivity at any of the dilutionsin the above range.

In an ELISA assay, the product to be screened is preferably used as thecoating antigen. A purified preparation is preferred, although a wholecell extract can also be used. Briefly, about 100 μl of a preparation atabout 10 μg protein/ml are distributed into wells of a 96-wellpolycarbonate ELISA plate. The plate is incubated for 2 hours at 37° C.then overnight at 4° C. The plate is washed with phosphate buffer saline(PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells aresaturated with 250 μl PBS containing 1% bovine serum albumin (BSA) toprevent non-specific antibody binding. After 1 hour incubation at 37°C., the plate is washed with PBS/Tween buffer. The antiserum is seriallydiluted in PBS/Tween buffer containing 0.5% BSA. 100 μl of dilutions areadded per well. The plate is incubated for 90 minutes at 37° C., washedand evaluated according to standard procedures. For example, a goatanti-rabbit peroxidase conjugate is added to the wells when specificantibodies were raised in rabbits. Incubation is carried out for 90minutes at 37° C. and the plate is washed. The reaction is developedwith the appropriate substrate and the reaction is measured bycolorimetry (absorbance measured spectrophotometrically). Under theabove experimental conditions, a positive reaction is shown by O.D.values greater than a non immune control serum.

In a dot blot assay, a purified product is preferred, although a wholecell extract can also be used. Briefly, a solution of the product atabout 100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5).100 μl of each dilution are applied to a nitrocellulose membrane 0.45 μmset in a 96-well dot blot apparatus (Biorad). The buffer is removed byapplying vacuum to the system. Wells are washed by addition of 50 mMTris-HCl (pH 7.5) and the membrane is air-dried. The membrane issaturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10g/L skim milk) and incubated with an antiserum dilution from about 1:50to about 1:5000, preferably about 1:500. The reaction is revealedaccording to standard procedures. For example, a goat anti-rabbitperoxidase conjugate is added to the wells when rabbit antibodies areused. Incubation is carried out 90 minutes at 37° C. and the blot iswashed. The reaction is developed with the appropriate substrate andstopped. The reaction is measured visually by the appearance of acolored spot, e.g., by colorimetry. Under the above experimentalconditions, a positive reaction is shown once a colored spot isassociated with a dilution of at least about 1:5, preferably of at leastabout 1:500.

Therapeutic or prophylactic efficacy of a polypeptide or derivative ofthe invention can be evaluated as described below. A seventh aspect ofthe invention provides (i) a composition of matter comprising apolypeptide of the invention together with a diluent or carrier;specifically (ii) a pharmaceutical composition containing atherapeutically or prophylactically effective amount of a polypeptide ofthe invention; (iii) a method for inducing an immune response againstChlamydia in a mammal, by administering to the mammal an immunogenicallyeffective amount of a polypeptide of the invention to elicit aprotective immune response to Chlamydia; and particularly, (iv) a methodfor preventing and/or treating a Chlamydia (e.g., C. trachomatis. C.psittaci, C. pneumoniae. or C. pecorum) infection, by administering aprophylactic or therapeutic amount of a polypeptide of the invention toan infected individual. Additionally, the seventh aspect of theinvention encompasses the use of a polypeptide of the invention in thepreparation of a medicament for preventing and/or treating Chlamydiainfection.

As used herein, the immunogenic compositions of the invention areadministered by conventional routes known the vaccine field, inparticular to a mucosal (e.g., ocular, intranasal, pulmonary, oral,gastric, intestinal, rectal, vaginal, or urinary tract) surface or viathe parenteral (e.g., subcutaneous, intradermal, intramuscular,intravenous, or intraperitoneal) route. The choice of administrationroute depends upon a number of parameters, such as the adjuvantassociated with the polypeptide. If a mucosal adjuvant is used, theintranasal or oral route is preferred. If a lipid formulation or analuminum compound is used, the parenteral route is preferred with thesub-cutaneous or intramuscular route being most preferred. The choicealso depends upon the nature of the vaccine agent. For example, apolypeptide of the invention fused to CTB or LTB is best administered toa mucosal surface.

As used herein, the composition of the invention contains one or severalpolypeptides or derivatives of the invention. The composition optionallycontains at least one additional Chlamydia antigen, or a subunit,fragment, homolog, mutant, or derivative thereof.

For use in a composition of the invention, a polypeptide or derivativethereof is formulated into or with liposomes, preferably neutral oranionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs)to facilitate delivery and/or enhance the immune response. Thesecompounds are readily available to one skilled in the art; for example,see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990).

Adjuvants other than liposomes and the like are also used and are knownin the art. Adjuvants may protect the antigen from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. An appropriateselection can conventionally be made by those skilled in the art, forexample, from those described below (under the eleventh aspect of theinvention).

Treatment is achieved in a single dose or repeated as necessary atintervals, as can be determined readily by one skilled in the art. Forexample, a priming dose is followed by three booster doses at weekly ormonthly intervals. An appropriate dose depends on various parametersincluding the recipient (e.g., adult or infant), the particular vaccineantigen, the route and frequency of administration, the presence/absenceor type of adjuvant, and the desired effect (e.g., protection and/ortreatment), as can be determined by one skilled in the art. In general,a vaccine antigen of the invention is administered by a mucosal route inan amount from about 10 μg to about 500 mg, preferably from about 1 mgto about 200 mg. For the parenteral route of administration, the doseusually does not exceed about 1 mg, preferably about 100 μg.

When used as vaccine agents, polynucleotides and polypeptides of theinvention may be used sequentially as part of a multistep immunizationprocess. For example, a mammal is initially primed with a vaccine vectorof the invention such as a pox virus, e.g., via the parenteral route,and then boosted twice with the polypeptide encoded by the vaccinevector, e.g., via the mucosal route. In another example, liposomesassociated with a polypeptide or derivative of the invention is alsoused for priming, with boosting being carried out mucosally using asoluble polypeptide or derivative of the invention in combination with amucosal adjuvant (e.g., LT).

A polypeptide derivative of the invention is also used in accordancewith the seventh aspect as a diagnostic reagent for detecting thepresence of anti-Chlamydia antibodies, e.g., in a blood sample. Suchpolypeptides are about 5 to about 80, preferably about 10 to about 50amino acids in length. They are either labeled or unlabeled, dependingupon the diagnostic method. Diagnostic methods involving such a reagentare described below.

Upon expression of a DNA molecule of the invention, a polypeptide orpolypeptide derivative is produced and purified using known laboratorytechniques. As described above, the polypeptide or polypeptidederivative may be produced as a fusion protein containing a fused tailthat facilitates purification. The fusion product is used to immunize asmall mammal, e.g., a mouse or a rabbit, in order to raise antibodiesagainst the polypeptide or polypeptide derivative (monospecificantibodies). Accordingly, an eighth aspect of the invention provides amonospecific antibody that binds to a polypeptide or polypeptidederivative of the invention.

By “monospecific antibody” is meant an antibody that is capable ofreacting with a unique naturally-occurring Chlamydia polypeptide. Anantibody of the invention is either polyclonal or monoclonal.Monospecific antibodies may be recombinant, e.g., chimeric (e.g.,constituted by a variable region of murine origin associated with ahuman constant region), humanized (a human immunoglobulin constantbackbone together with hypervariable region of animal, e.g., murine,origin), and/or single chain. Both polyclonal and monospecificantibodies may also be in the form of immunoglobulin fragments, e.g.,F(ab)′2 or Fab fragments. The antibodies of the invention are of anyisotype, e.g., IgG or IgA, and polyclonal antibodies are of a singleisotype or a mixture of isotypes.

Antibodies against the polypeptides, homologs or fragments of thepresent invention are generated by immunization of a mammal with acomposition comprising said polypeptide, homolog or fragment. Suchantibodies may be polyclonal or monoclonal. Methods to producepolyclonal or monoclonal antibodies are well known in the art. For areview, see “Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al.,1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, seeKohler & Milstein (1975) Nature 256:495-497.

The antibodies of the invention, which are raised to a polypeptide orpolypeptide derivative of the invention, are produced and identifiedusing standard immunological assays, e.g., Western blot analysis, dotblot assay, or ELISA (see, e.g., Coligan et al., Current Protocols inImmunology (1994) John Wiley & Sons, Inc., New York, N.Y.). Theantibodies are used in diagnostic methods to detect the presence of aChlamydia antigen in a sample, such as a biological sample. Theantibodies are also used in affinity chromatography for purifying apolypeptide or polypeptide derivative of the invention. As is discussedfurther below, such antibodies may be used in prophylactic andtherapeutic passive immunization methods.

Accordingly, a ninth aspect of the invention provides (i) a reagent fordetecting the presence of Chlamydia in a biological sample that containsan antibody, polypeptide, or polypeptide derivative of the invention;and (ii) a diagnostic method for detecting the presence of Chlamydia ina biological sample, by contacting the biological sample with anantibody, a polypeptide, or a polypeptide derivative of the invention,such that an immune complex is formed, and by detecting such complex toindicate the presence of Chiamydia in the sample or the organism fromwhich the sample is derived.

Those skilled in the art will readily understand that the immune complexis formed between a component of the sample and the antibody,polypeptide, or polypeptide derivative, whichever is used, and that anyunbound material is removed prior to detecting the complex. It isunderstood that a polypeptide reagent is useful for detecting thepresence of anti-Chlamydia antibodies in a sample, e.g., a blood sample,while an antibody of the invention is used for screening a sample, suchas a gastric extract or biopsy, for the presence of Chlamydiapolypeptides.

For diagnostic applications, the reagent (i.e., the antibody,polypeptide, or polypeptide derivative of the invention) is either in afree state or immobilized on a solid support, such as a tube, a bead, orany other conventional support used in the field. Immobilization isachieved using direct or indirect means. Direct means include passiveadsorption (non-covalent binding) or covalent binding between thesupport and the reagent. By “indirect means” is meant that ananti-reagent compound that interacts with a reagent is first attached tothe solid support. For example, if a polypeptide reagent is used, anantibody that binds to it can serve as an anti-reagent, provided that itbinds to an epitope that is not involved in the recognition ofantibodies in biological samples. Indirect means may also employ aligand-receptor system, for example, where a molecule such as a vitaminis grafted onto the polypeptide reagent and the corresponding receptorimmobilized on the solid phase. This is illustrated by thebiotin-streptavidin system. Alternatively, a peptide tail is addedchemically or by genetic engineering to the reagent and the grafted orfused product immobilized by passive adsorption or covalent linkage ofthe peptide tail.

Such diagnostic agents may be included in a kit which also comprisesinstructions for use. The reagent is labeled with a detection meanswhich allows for the detection of the reagent when it is bound to itstarget. The detection means may be a fluorescent agent such asfluorescein isocyanate or fluorescein isothiocyanate, or an enzyme suchas horse radish peroxidase or luciferase or alkaline phosphatase, or aradioactive element such as ¹²⁵I or ⁵¹Cr.

Accordingly, a tenth aspect of the invention provides a process forpurifying, from a biological sample, a polypeptide or polypeptidederivative of the invention, which involves carrying out antibody-basedaffinity chromatography with the biological sample, wherein the antibodyis a monospecific antibody of the invention.

For use in a purification process of the invention, the antibody iseither polyclonal or monospecific, and preferably is of the IgG type.Purified IgGs is prepared from an antiserum using standard methods (see,e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley& Sons, Inc., New York, N.Y.). Conventional chromatography supports, aswell as standard methods for grafting antibodies, are described in,e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988)and outlined below.

Briefly, a biological sample, such as an C. pneumoniae extractpreferably in a buffer solution, is applied to a chromatographymaterial, preferably equilibrated with the buffer used to dilute thebiological sample so that the polypeptide or polypeptide derivative ofthe invention (i.e., the antigen) is allowed to adsorb onto thematerial. The chromatography material, such as a gel or a resin coupledto an antibody of the invention, is in either a batch form or a column.The unbound components are washed off and the antigen is then elutedwith an appropriate elution buffer, such as a glycine buffer or a buffercontaining a chaotropic agent, e.g., guanidine HCl, or high saltconcentration (e.g., 3 M MgCl₂). Eluted fractions are recovered and thepresence of the antigen is detected, e.g., by measuring the absorbanceat 280 nm.

An eleventh aspect of the invention provides (i) a composition of mattercomprising a monospecific antibody of the invention, together with adiluent or carrier; (ii) a pharmaceutical composition comprising atherapeutically or prophylactically effective amount of a monospecificantibody of the invention, and (iii) a method for treating or preventinga Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C.pecorum) infection, by administering a therapeutic or prophylacticamount of a monospecific antibody of the invention to an infectedindividual. Additionally, the eleventh aspect of the inventionencompasses the use of a monospecific antibody of the invention in thepreparation of a medicament for treating or preventing Chlamydiainfection.

The monospecific antibody is either polyclonal or monoclonal, preferablyof the IgA isotype (predominantly). In passive immunization, theantibody is administered to a mucosal surface of a mammal, e.g., thegastric mucosa, e.g., orally or intragastrically, advantageously, in thepresence of a bicarbonate buffer. Alternatively, systemicadministration, not requiring a bicarbonate buffer, is carried out. Amonospecific antibody of the invention is administered as a singleactive component or as a mixture with at least one monospecific antibodyspecific for a different Chlamydia polypeptide. The amount of antibodyand the particular regimen used are readily determined by one skilled inthe art. For example, daily administration of about 100 to 1,000 mg ofantibodies over one week, or three doses per day of about 100 to 1,000mg of antibodies over two or three days, are effective regimens for mostpurposes.

Therapeutic or prophylactic efficacy are evaluated using standardmethods in the art, e.g., by measuring induction of a mucosal immuneresponse or induction of protective and/or therapeutic immunity, using,e.g., the C. pneumoniae mouse model. Those skilled in the art willreadily recognize that the C. pneumoniae strain of the model may bereplaced with another Chlamydia strain. For example, the efficacy of DNAmolecules and polypeptides from C. pneumoniae is preferably evaluated ina mouse model using C. pneumoniae strain. Protection is determined bycomparing the degree of Chlamydia infection to that of a control group.Protection is shown when infection is reduced by comparison to thecontrol group. Such an evaluation is made for polynucleotides, vaccinevectors, polypeptides and derivatives thereof, as well as antibodies ofthe invention.

Adjuvants useful in any of the vaccine compositions described above areas follows.

Adjuvants for parenteral administration include aluminum compounds, suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate. The antigen is precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants, suchas RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteraladministration.

Adjuvants for mucosal administration include bacterial toxins, e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof such as a purified preparation ofnative cholera toxin subunit B (CTB). Fragments, homologs, derivatives,and fusions to any of these toxins are also suitable, provided that theyretain adjuvant activity. Preferably, a mutant having reduced toxicityis used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-LysCT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323(Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that areused in the methods and compositions of the invention include, e.g.,Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Otheradjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g.,E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigellaflexneri; saponins, or polylactide glycolide (PLGA) microspheres, isalso be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral administrations includepolyphosphazene (WO 95/02415), DC-chol (3 b-(N-(N′,N′-dimethylaminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and WO96/14831) and QS-21 (WO 88/09336).

Any pharmaceutical composition of the invention containing apolynucleotide, a polypeptide, a polypeptide derivative, or an antibodyof the invention, is manufactured in a conventional manner. Inparticular, it is formulated with a pharmaceutically acceptable diluentor carrier, e.g., water or a saline solution such as phosphate buffersaline. In general, a diluent or carrier is selected on the basis of themode and route of administration, and standard pharmaceutical practice.Suitable pharmaceutical carriers or diluents, as well as pharmaceuticalnecessities for their use in pharmaceutical formulations, are describedin Remington's Pharmaceutical Sciences, a standard reference text inthis field and in the USP/NF.

The invention also includes methods in which Chlamydia infection aretreated by oral administration of a Chlamydia polypeptide of theinvention and a mucosal adjuvant, in combination with an antibiotic, anantacid, sucralfate, or a combination thereof. Examples of suchcompounds that can be administered with the vaccine antigen and theadjuvant are antibiotics, including, e.g., macrolides, tetracyclines,and derivatives thereof (specific examples of antibiotics that can beused include azithromycin or doxicyclin or immunomodulators such ascytokines or steroids). In addition, compounds containing more than oneof the above-listed components coupled together, are used. The inventionalso includes compositions for carrying out these methods, i.e.,compositions containing a Chlamydia antigen (or antigens) of theinvention, an adjuvant, and one or more of the above-listed compounds,in a pharmaceutically acceptable carrier or diluent.

It has recently been shown that the 60 kDa cysteine rich membraneprotein contains a sequence cross-reactive with the murine alpha-myosinheavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaieret al., Science (1999) 283:1335). This cross-reactivity is proposed tocontribute to the development of cardiovascular disease, so it may bebeneficial to remove this epitope, and any other epitopes cross-reactivewith human antigens, from the protein if it is to be used as a vaccine.Accordingly, a further embodiment of the present invention includes themodification of the coding sequence, for example, by deletion orsubstitution of the nucleotides encoding the epitope frompolynucleotides encoding the protein, as to improve the efficacy andsafety of the protein as a vaccine. A similar approach may beappropriate for any protective antigen found to have unwanted homologiesor cross-reactivities with human antigens.

Amounts of the above-listed compounds used in the methods andcompositions of the invention are readily determined by one skilled inthe art. Treatment/immunization schedules are also known and readilydesigned by one skilled in the art. For example, the non-vaccinecomponents can be administered on days 1-14, and the vaccineantigen+adjuvant can be administered on days 7, 14, 21, and 28.

EXAMPLES

The above disclosure generally discribes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Example 1

This example illustrates the preparation of a plasmid vector pCACRMP60containing the 60 kDa cysteine rich membrane protein gene.

The 60 kDa cysteine rich membrane protein gene was amplified fromChlamydia pneumoniae genomic DNA by polymerase chain reaction (PCR)using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGTCCAAACTCATCAGACGAGTAG3′)(SEQ ID No:3) and a 3′ primer (5′ GCGCCGGATCCGATACACGTGGGTATTTTCTGTG3′) (SEQ ID No:4). The 5′ primer contains a Not I restriction site, aribosome binding site, an initiation codon and a sequence at the 5′ endof the 60 kDa cysteine rich membrane protein coding sequence. The 3′primer includes the sequence encoding the C-terminal sequence of the 60kDa cysteine rich membrane protein and a Bam HI restriction site. Thestop codon was excluded and an additional nucleotide was inserted toobtain an in-frame fusion with the Histidine tag.

After amplification, the PCR fragment was purified using QIAquick™ PCRpurification kit (Qiagen) and then digested with Not I and Bam HI andcloned into the pCA-Myc-His eukaryotic expression vector describe inexample 2 (FIG. 3) with transcription under control of the human CMVpromoter.

Example 2

This example illustrates the preparation of the eukaryotic expressionvector pCA/Myc-His.

Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with Spe I andBam HI to remove the CMV promoter and the remaining vector fragment wasisolated. The CMV promoter and intron A from plasmid VR-1012 (Vical) wasisolated on a Spe I/Bam HI fragment. The fragments were ligated togetherto produce plasmid pCA/Myc-His. The Not I/Bam HI restricted PCR fragmentcontaining the 60 kDa cysteine rich membrane protein gene was ligatedinto the Not I and Bam HI restricted plasmid pCA/Myc-His to produceplasmid pCACRMP60 (FIG. 3).

The resulting plasmid, pCACRMP60, was transferred by electroporationinto E. coli XL-1 blue (Stratagene) which was grown in LB brothcontaining 50 μg/ml of carbenicillin. The plasmid was isolated by EndoFree Plasmid Giga Kit™ (Qiagen) large scale DNA purification system. DNAconcentration was determined by absorbance at 260 nm and the plasmid wasverified after gel electrophoresis and Ethidium bromide staining andcomparison to molecular weight standards. The 5′ and 3′ ends of the genewere verified by sequencing using a LiCor model 4000 L DNA sequencer andIRD-800 labelled primers.

Example 3

This example illustrates the immunization of mice to achieve protectionagainst an intranasal challenge of C. pneumoniae.

It has been previously demonstrated (Yang et. al., 1993) that mice aresusceptible to intranasal infection with different isolates of C.pneumoniae. Strain AR-39 (Grayston, 1989) was used in Balb/c mice as achallenge infection model to examine the capacity of chlamydia geneproducts delivered as naked DNA to elicit a protective response againsta sublethal C. pneumoniae lung infection. Protective immunity is definedas an accelerated clearance of pulmonary infection.

Groups of 7 to 9 week old male Balb/c mice (8 to 10 per group) wereimmunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmidDNA containing the coding sequence of C. pneumoniae 60 kDa cysteine richmembrane protein as described in Example 1 and 2. Saline or the plasmidvector lacking an inserted chlamydial gene was given to groups ofcontrol animals.

For i.m. immunization alternate left and right quadriceps were injectedwith 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3 and 6weeks. For i.n. immunization, anaesthetized mice aspirated 50 μl of PBScontaining 50 μg DNA on three occasions at 0, 3 and 6 weeks. At week 8,immunized mice were inoculated i.n. with 5×10⁵ IFU of C. pneumoniae,strain AR39 in 10011 of SPG buffer to test their ability to limit thegrowth of a sublethal C. pneumoniae challenge.

Lungs were taken from mice at days 5 and 9 post-challenge andimmediately homogenised in SPG buffer (7.5% sucrose, 5 mM glutamate,12.5 mM phosphate pH7.5). The homogenate was stored frozen at −70° C.until assay. Dilutions of the homogenate were assayed for the presenceof infectious chlamydia by inoculation onto monolayers of susceptiblecells. The inoculum was centrifuged onto the cells at 3000 rpm for 1hour, then the cells were incubated for three days at 35° C. in thepresence of 1 μg/ml cycloheximide. After incubation the monolayers werefixed with formalin and methanol then immunoperoxidase stained for thepresence of chlamydial inclusions using convalescent sera from rabbitsinfected with C. pneumoniae and metal-enhanced DAB as a peroxidasesubstrate.

FIG. 4 and Table 1 show that mice immunized i.n. and i.m. with pCACRMP60had chlamydial lung titers less than 1400 in 4 of 4 cases at day 5 andless than 400 in 4 of 4 cases at day 9 whereas the range of values forcontrol mice sham immunized with saline was 1200-2100 IFU/lung (mean1680) at day 5 and 1100-12400 IFU/lung (mean 4320) at day 9. DNAimmunisation per se was not responsible for the observed protectiveeffect since another plasmid DNA construct, pCAI422, failed to protect,with lung titers in immunised mice similar to those obtained forsaline-immunized control mice. The construct pCAI422 is identical topCACRMP60 except that the nucleotide sequence encoding the 60 kDacysteine rich membrane protein is replaced with a C. pneumoniaenucleotide sequence encoding a non-protective protein. TABLE 1 BACTERIALLOAD (INCLUSION FORMING UNITS PER LUNG) IN THE LUNGS OF BALB/C MICEIMMUNIZED WITH VARIOUS DNA IMMUNIZATION CONSTRUCTS IMMUNIZING CONSTRUCTSaline Saline pCACRMP60 pCACRMP60 pCA1422 pCA1422 MOUSE Day 5 Day 9 Day5 Day 9 Day 5 Day 9 1 1800 1100 200 300 2200 3100 2 1200 2100 100 0 13008100 3 2100 12400 500 200 700 500 4 1900 3900 1300 0 1000 1400 5 14002100 MEAN 1680 4320 525 125 1300 3275 SD 370.14 4628.39 543.9056 150.00648.07 3392.52

1-37. (canceled)
 38. A vaccine vector comprising an isolated nucleic acid molecule which encodes SEQ ID No: 2, wherein the nucleic acid molecule is operably linked to a promoter functional in a mammalian cell.
 39. The vaccine vector of claim 38 further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide comprising SEQ ID No: 2, wherein the additional polypeptide is a Chlamydia polypeptide.
 40. The vaccine vector of claim 38 wherein the promoter is a viral promoter.
 41. The vaccine vector of claim 39 wherein the promoter is a viral promoter.
 42. The vaccine vector of claim 40 wherein the promoter is cytomegalovirus (CMV) promoter.
 43. The vaccine vector of claim 41 wherein the promoter is cytomegalovirus (CMV) promoter.
 44. The vaccine vector of claim 38 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 45. The vaccine vector of claim 39 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 46. The vaccine vector of claim 40 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 47. The vaccine vector of claim 41 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 48. The vaccine vector of claim 42 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 49. The vaccine vector of claim 43 wherein the nucleotide sequence of the nucleic acid molecule which encodes SEQ ID No:2 is SEQ ID No:1.
 50. A vaccine comprising the vaccine vector according to claim 38 and a pharmaceutically acceptable carrier.
 51. A vaccine comprising the vaccine vector according to claim 39 and a pharmaceutically acceptable carrier.
 52. A vaccine comprising the vaccine vector according to claim 40 and a pharmaceutically acceptable carrier.
 53. A vaccine comprising the vaccine vector according to claim 41 and a pharmaceutically acceptable carrier.
 54. A vaccine comprising the vaccine vector according to claim 42 and a pharmaceutically acceptable carrier.
 55. A vaccine comprising the vaccine vector according to claim 43 and a pharmaceutically acceptable carrier.
 56. A vaccine comprising the vaccine vector according to claim 44 and a pharmaceutically acceptable carrier.
 57. A vaccine comprising the vaccine vector according to claim 45 and a pharmaceutically acceptable carrier. 